Purified PH neutral Rhizoctonia laccases and nucleic acids encoding same

Abstract

The present invention relates to isolated nucleic acid fragments containing a sequence encoding a Rhizoctonia solani laccase having optimum activity at a neutral or basic pH. and the laccase proteins encoded thereby.

What we claim is: 
     
       1. An isolated nucleic acid fragment containing a nucleic acid sequence encoding a Rhizoctonia solani laccase which functions optimally at a pH between about 6.0 and 8.5. 
     
     
       2. The fragment of claim 1 which comprises a nucleic acid sequence encoding the amino acid sequence depicted in SEQ ID NO. 2. 
     
     
       3. The fragment of claim 1 which comprises a nucleic acid sequence encoding the amino acid sequence depicted in SEQ ID NO. 4. 
     
     
       4. The fragment of claim 1, which comprises a nucleic acid sequence encoding a protein containing one or more of the amino acid sequences depicted in SEQ. ID NOS. 5, 6, 7, 8, 9, 10, 11, or 12. 
     
     
       5. The fragment of claim 1 which comprises a nucleic acid sequence encoding the amino acid sequence depicted in SEQ ID NO. 14. 
     
     
       6. The fragment of claim 1, which comprises the nucleic acid sequence depicted in SEQ ID NO. 1). 
     
     
       7. The fragment of claim 1, which comprises the nucleic acid sequence depicted in SEQ. ID. NO. 3. 
     
     
       8. The fragment of claim 1, which comprises the nucleic acid sequence depicted in SEQ. ID. NO. 13. 
     
     
       9. The fragment of claim 1, which comprises the nucleic acid sequence contained in NRRL B-21141. 
     
     
       10. The fragment of claim 1, which comprises the nucleic acid sequence contained in NRRL B-21142. 
     
     
       11. The fragment of claim 1, which comprises the nucleic acid sequence encoding the laccase produced by RS 22. 
     
     
       12. The fragment of claim 1, which comprises the nucleic acid sequence contained in NRRL B-21156. 
     
     
       13. A recombinant vector comprising a nucleic acid fragment of claim 1. 
     
     
       14. The vector of claim 13 in which the fragment is operably linked to a promoter sequence. 
     
     
       15. The vector of claim 14 in which the promoter is a fungal or yeast promoter. 
     
     
       16. The vector of claim 15 in which the promoter is the TAKA amylase promoter of Aspergillus oryzae. 
     
     
       17. The vector of claim 15 in which the promoter is the glucoamylase (gluA) promoter of Aspergillus niger or Aspergillus awamsii. 
     
     
       18. The vector of claim 14 which also comprises a selectable marker. 
     
     
       19. The vector of claim 18 in which the selectable marker is the amdS marker of Aspergillus nidulans or Aspergillus oryzae. 
     
     
       20. The vector of claim 18 in which the selectable marker is the pyrG marker cf Aspergillus nidulans, Aspergillus niger, Aspergillus awamsii, or Aspergillus oryzae. 
     
     
       21. The vector of claim 14 which comprises both the TAKA amylase promoter of Aspergillus oryzae and the amdS or pyrG marker of Aspergillus nidulans or Aspergillus oryzae. 
     
     
       22. A host cell comprising a heterologous nucleic acid fragment of claim 1, 
     
     
       23. The host cell of claim 22 which is a fungal cell. 
     
     
       24. The host cell of claim 23 which is an Aspergillus cell. 
     
     
       25. The host cell of claim 22 in which the fragment is integrated into the host cell genome. 
     
     
       26. The host cell of claim 22 in which the fragment is contained on a vector. 
     
     
       27. The host cell of claim 22 which comprises a fragment containing a sequence encoding the amino acid sequence depicted in SEQ ID NO. 2. 
     
     
       28. The host cell of claim 22 which comprises a fragment containing a sequence encoding the amino acid sequence depicted in SEQ ID NO: 4. 
     
     
       29. The host cell of claim 22 which comprises a fragment containing a sequence encoding the amino acid sequence depicted in SEQ ID NO: 14. 
     
     
       30. The host cell of claim 22 which comprises a fragment containing a sequence encoding one or more of the amino acid sequences depicted in SEQ ID NOS.: 5, 6, 7, 8, 9, 10, 11, or 12.